Variation in mate-recognition pheromones of the fungal genus Microbotryum.

Mate recognition is an essential life-cycle stage that exhibits strong conservation in function, whereas diversification of mating signals can contribute directly to the integrity of species boundaries through assortative mating. Fungi are simple models, where compatibility is based on the recognition of pheromone peptides by corresponding receptor proteins, but clear patterns of diversification have not emerged from the species examined, which are few compared with mate signaling studies in plant and animal systems. In this study, candidate loci from Microbotryum species were used to characterize putative pheromones that were synthesized and found to be functional across multiple species in triggering a mating response in vitro. There is no significant correlation between the strength of a species' response and its genetic distance from the pheromone sequence source genome. Instead, evidence suggests that species may be strong or weak responders, influenced by environmental conditions or developmental differences. Gene sequence comparisons reveals very strong purifying selection on the a1 pheromone peptide and corresponding receptor, but significantly less purifying selection on the a2 pheromone peptide that corresponds with more variation across species in the receptor. This represents an exceptional case of a reciprocally interacting mate-recognition system in which the two mating types are under different levels of purifying selection.

The population biology of fungal invasions.

Fungal invasions are increasingly recognized as a significant component of global changes, threatening ecosystem health and damaging food production. Invasive fungi also provide excellent models to evaluate the generality of results based on other eukaryotes. We first consider here the reasons why fungal invasions have long been overlooked: they tend to be inconspicuous, and inappropriate methods have been used for species recognition. We then review the information available on the patterns and mechanisms of fungal invasions. We examine the biological features underlying invasion success of certain fungal species. We review population structure analyses, revealing native source populations and strengths of bottlenecks. We highlight the documented ecological and evolutionary changes in invaded regions, including adaptation to temperature, increased virulence, hybridization, shifts to clonality and association with novel hosts. We discuss how the huge census size of most fungi allows adaptation even in bottlenecked, clonal invaders. We also present new analyses of the invasion of the anther-smut pathogen on white campion in North America, as a case study illustrating how an accurate knowledge of species limits and phylogeography of fungal populations can be used to decipher the origin of invasions. This case study shows that successful invasions can occur even when life history traits are particularly unfavourable to long-distance dispersal and even with a strong bottleneck. We conclude that fungal invasions are valuable models to contribute to our view of biological invasions, in particular by providing insights into the traits as well as ecological and evolutionary processes allowing successful introductions.

Evolution of uni- and bifactorial sexual compatibility systems in fungi.

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.

Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution.

Hosts and their symbionts are involved in intimate physiological and ecological interactions. The impact of these interactions on the evolution of each partner depends on the time-scale considered. Short-term dynamics - 'coevolution' in the narrow sense - has been reviewed elsewhere. We focus here on the long-term evolutionary dynamics of cospeciation and speciation following host shifts. Whether hosts and their symbionts speciate in parallel, by cospeciation, or through host shifts, is a key issue in host-symbiont evolution. In this review, we first outline approaches to compare divergence between pairwise associated groups of species, their advantages and pitfalls. We then consider recent insights into the long-term evolution of host-parasite and host-mutualist associations by critically reviewing the literature. We show that convincing cases of cospeciation are rare (7%) and that cophylogenetic methods overestimate the occurrence of such events. Finally, we examine the relationships between short-term coevolutionary dynamics and long-term patterns of diversification in host-symbiont associations. We review theoretical and experimental studies showing that short-term dynamics can foster parasite specialization, but that these events can occur following host shifts and do not necessarily involve cospeciation. Overall, there is now substantial evidence to suggest that coevolutionary dynamics of hosts and parasites do not favor long-term cospeciation.

Sibling competition arena: selfing and a competition arena can combine to constitute a barrier to gene flow in sympatry.

Closely related species coexisting in sympatry provide critical insight into the mechanisms underlying speciation and the maintenance of genetic divergence. Selfing may promote reproductive isolation by facilitating local adaptation, causing reduced hybrid fitness in parental environments. Here, we propose a novel mechanism by which selfing can further impair interspecific gene flow: selfing may act to ensure that nonhybrid progeny systematically co-occur whenever hybrid genotypes are produced. Under a competition arena, the fitness differentials between nonhybrid and hybrid progeny are then magnified, preventing development of interspecific hybrids. We investigate whether this "sibling competition arena" can explain the coexistence in sympatry of closely related species of the plant fungal pathogens (Microbotryum) causing anther-smut disease. The probabilities of intrapromycelial mating (automixis), outcrossing, and sibling competition were manipulated in artificial inoculations to evaluate their contribution to reproductive isolation. We report that both intrapromycelial selfing and sibling competition significantly reduced rates of hybrid infection beyond that expected based solely upon selfing rates and noncompetitive fitness differentials between hybrid and nonhybrid progeny. Our results thus suggest that selfing and a sibling competition arena can combine to constitute a barrier to gene flow and diminish selection for additional barriers to gene flow in sympatry.

Sex, outcrossing and mating types: unsolved questions in fungi and beyond.

Variability in the way organisms reproduce raises numerous, and still unsolved, questions in evolutionary biology. In this study, we emphasize that fungi deserve a much greater emphasis in efforts to address these questions because of their multiple advantages as model eukaryotes. A tremendous diversity of reproductive modes and mating systems can be found in fungi, with many evolutionary transitions among closely related species. In addition, fungi show some peculiarities in their mating systems that have received little attention so far, despite the potential for providing insights into important evolutionary questions. In particular, selfing can occur at the haploid stage in addition to the diploid stage in many fungi, which is generally not possible in animals and plants but has a dramatic influence upon the structure of genetic systems. Fungi also present several advantages that make them tractable models for studies in experimental evolution. Here, we briefly review the unsolved questions and extant hypotheses about the evolution and maintenance of asexual vs. sexual reproduction and of selfing vs. outcrossing, focusing on fungal life cycles. We then propose how fungi can be used to address these long-standing questions and advance our understanding of sexual reproduction and mating systems across all eukaryotes.

Dynamic linkage relationships to the mating-type locus in automictic fungi of the genus Microbotryum.

Regions of the chromosomes determining mating compatibility in some fungi, including Microbotryum lychnidis-dioicae and Neurospora tetrasperma, exhibit suppressed recombination similar to sex chromosomes in plants and animals, and recent studies have sought to apply basic theories of sex chromosome evolution to fungi. A phylogeny of the MTL1 locus in Microbotryum indicates that it has become part of the nonrecombining regions of the mating-type chromosomes in multiple independent events, and that recombination may have been subsequently restored in some cases. This illustrates that fungal mating-type chromosomes can exhibit linkage relationship that are quite dynamic, adding to the list of similarities to animal or plant sex chromosomes. However, fungi such as M. lychnidis-dioicae and N. tetrasperma exhibit an automictic mating system, for which an alternate theoretical framework exists to explain the evolution of linkage with the mating-type locus. This study encourages further comparative studies among fungi to evaluate the role of mating systems in determining the evolution of fungal mating-type chromosomes.

Phylogenetic determinants of potential host shifts in fungal pathogens.

Understanding what determines the host range of pathogens and the potential for host shifts is of critical importance to controlling their introductions into new environments. The phylogeny of the hosts has been shown to be important: pathogens are more likely to be infectious on hosts closely related to their host-of-origin because of the similar host environments that is shared by descent. The importance of pathogen phylogenies for predicting host range has never been investigated, although a pathogen should also be able to exploit a new host that its close relative can infect. We performed cross-inoculations using a plant-fungal association and showed that both host and pathogen phylogenies were significant predictors of host range, with at least partly independent effects. Furthermore, we showed that some pathogens were better at infecting novel hosts. Our results should have implications in the context of biological invasions and emergences of new diseases due to globalization.

Silene as a model system in ecology and evolution.

The genus Silene, studied by Darwin, Mendel and other early scientists, is re-emerging as a system for studying interrelated questions in ecology, evolution and developmental biology. These questions include sex chromosome evolution, epigenetic control of sex expression, genomic conflict and speciation. Its well-studied interactions with the pathogen Microbotryum has made Silene a model for the evolution and dynamics of disease in natural systems, and its interactions with herbivores have increased our understanding of multi-trophic ecological processes and the evolution of invasiveness. Molecular tools are now providing new approaches to many of these classical yet unresolved problems, and new progress is being made through combining phylogenetic, genomic and molecular evolutionary studies with ecological and phenotypic data.

Hybrid sterility and inviability in the parasitic fungal species complex Microbotryum.

Microbotryum violaceum, the anther-smut fungus, forms a complex of sibling species which specialize on different plants. Previous studies have shown the presence of partial ecological isolation and F1 inviability, but did not detect assortative mating apart from a high selfing rate. We investigated other post-mating barriers and show that F1 hybrid sterility, the inability of gametes to mate, increased gradually with the increasing genetic distance between the parents. F2 hybrids showed a reduced ability to infect the plants that was also correlated with the genetic distance. The host on which the F2 hybrids were passaged caused a selection for alleles derived from the pathogen species originally isolated from that host, but this effect was not detectable for the most closely related species. The post-mating barriers thus remain weak among the closest species pairs, suggesting that premating barriers are sufficient to initiate divergence in this system.

Maximized virulence in a sterilizing pathogen: the anther-smut fungus and its co-evolved hosts.

Host sterilization is a common feature of sexually transmitted diseases (STDs). Because host reproductive failure may free up resources for pathogen reproduction and transmission, theory predicts that selection on sterilizing pathogens will favour maximum virulence (i.e. complete sterilization). We examined patterns of infection in sexually transmitted anther-smut fungi (Microbotryum) on four of their host species in the Caryophyllaceae. Using controlled fungal matings and experimental inoculations, we compared disease expression in inoculations ranging from host-specific pathogens to hybrids and cross-species treatments. Our data support the existence of host-specific sibling species within the genus Microbotryum based on a low infection rate from cross-inoculations and reduced fitness for hybrid pathogens. These patterns of host specificity and reproductive isolation, however, were not absolute. We did observe some successful cross-species and hybrid infections, but the expression of disease was frequently incomplete, including only partial host sterilization and the failed dehiscence of pathogen spores. The prevalence of these maladapted disease phenotypes may greatly inhibit the emergence of novel host pathogen combinations. Infections by hybrid pathogen genotypes were intermediate, in terms of both infection rate and the normality of disease symptoms, between host-specific and cross-inoculated pathogens. In addition, the frequency with which hybrid and cross-inoculated anther-smut pathogens were able to infect but not sterilize new hosts supports the prediction that sterilizing STDs are under selection to maximize virulence in natural populations.

PERMANENT GENETIC RESOURCES: Isolation of 60 polymorphic microsatellite loci in EST libraries of four sibling species of the phytopathogenic fungal complex Microbotryum.

We report the development of 60 microsatellite markers on four species of the fungal complex Microbotryum, causing anther smut of the Caryophyllaceae. Microsatellites were found in four expressed sequence tag (EST) libraries, built from isolates of M. lychnis-dioicae, M. violaceum sensus stricto, M. lagerheimii and M. dianthorum, collected, respectively, from the plants Silene latifolia, S. nutans, S. vulgaris and Dianthus carthusianorum. Intrapopulation polymorphism was investigated using 24 isolates, and cross-amplification was explored using 23 isolates belonging to at least 10 different Microbotryum species. This study provides numerous microsatellite markers for population genetics and mapping studies.

Pathogen relatedness affects the prevalence of within-host competition.

Although the evolutionary consequences of within-host competition among pathogens have been examined extensively, there exists a critical gap in our understanding of factors determining the prevalence of multiple infections. Here we examine the effects of relatedness among strains of the anther-smut pathogen Microbotryum violaceum on the probability of multiple infection in its host, Silene latifolia, after sequential inoculations. We found a significantly higher probability of multiple infection when interacting strains were more closely related, suggesting mechanisms of competitive exclusion that are conditional on genotypic characteristics of the strains involved. Pathogen relatedness therefore determines the prevalence of multiple infection in addition to its outcome, with important consequences for our understanding of virulence evolution and pathogen population structure and diversity.

The anther smut disease on Gypsophila repens: a case of parasite sub-optimal performance following a recent host shift?

The study of how parasites adapt to new hosts is of great importance for understanding the emergence of new diseases. Here, we report a study of the anther smut disease on Gypsophila repens (Caryophyllaceae). In contrast to what is usually found on other host species, infected natural populations of G. repens are extremely rare. Moreover, symptoms of diseased plants are incomplete and highly variable over the time. These results suggest that the fungus infecting G. repens is a case of a parasite not capable of exploiting its host optimally. Molecular analyses of Microbotryum violaceum strains infecting this and other Caryophyllaceae revealed that this sub-optimal behaviour probably resulted from a recent host shift from the morphologically similar plant Petrorhagia saxifraga. With its exceptionally low virulence and prevalence, but apparent self-sustainability, the disease on G. repens may thus represent an interesting case study for investigating the conditions leading to adaptation of parasites on new hosts.

Dynamics of multiple infection and within-host competition by the anther-smut pathogen.

Infection of one host by multiple pathogen genotypes represents an important area of pathogen ecology and evolution that lacks a broad empirical foundation. Multiple infection of Silene latifolia by Microbotryum violaceum was studied under field and greenhouse conditions using the natural polymorphism for mating-type bias as a marker. Field transmission resulted in frequent multiple infection, and each stem of the host was infected independently. Within-host diversity of infections equaled that of nearby inoculum sources by the end of the growing season. The number of diseased stems per plant was positively correlated with multiple infection and with overwintering mortality. As a result, multiply infected plants were largely purged from the population, and there was lower within-host pathogen diversity in the second season. However, among plants with a given number of diseased stems, multiply infected plants had a lower risk of overwintering mortality. Following simultaneous and sequential inoculation, strong competitive exclusion was demonstrated, and the first infection had a significant advantage. Dynamics of multiple infection initially included components of coinfection models for virulence evolution and then components of superinfection models after systemic colonization. Furthermore, there was evidence for an advantage of genotypes with mating-type bias, which may contribute to maintenance of this polymorphism in natural populations.

Intratetrad mating, heterozygosity, and the maintenance of deleterious alleles in Microbotryum violaceum (=Ustilago violacea).

The mating system of Microbotryum violaceum was investigated in populations that are polymorphic for mating-type bias, where individuals produce viable haploids of only one of the two required mating types. The cause of mating-type bias was identified as deleterious recessive alleles linked to mating type. Maintenance of the deleterious alleles was promoted by early conjugation among products of single meioses, such that the duration of the free-living haploid stage is minimized. This development was also observed in nonbiased isolates. As a consequence, the mating system tends toward mating within the tetrad, which might be expected to reduce heterozygosity. However, complete centromere linkage of mating type ensures conjugation between first division meiotic products, such that mating in M. violaceum is analogous to forms of meiotic parthenogenesis with first division restitution (i.e. automixis with central fusion). This fungus was used to test the prediction that this mating system would maintain heterozygosity in regions of the genome linked to centromeres. Therefore, populations were screened for additional heterozygous lethal recessive alleles linked to centromeres, and several examples were found. Furthermore, the occurrence of intratetrad mating in M. violaceum provides an explanation for low variation among individuals within populations, inconsistent estimates of outcrossing rates, low levels of mating between tetrads of one diploid individual, and high frequencies of haplo-lethal alleles in natural populations.

Primary Haustorial Development of Striga asiatica on Host and Nonhost Species.

ABSTRACT Initial interactions of Striga asiatica with a susceptible host and non-host plants were examined by histological methods. Haustorial development was initiated when radicles of S. asiatica were placed in contact with host or nonhost roots. Reorganization of the S. asiatica root apical meristem was rapid and involved the formation of a distal group of cells that penetrated the host or nonhost root. Penetration of the epidermis of the host (sorghum) roots and advance into the cortex occurred within 24 to 48 h of inoculation. Penetration of the endodermis by the developing endophyte was delayed for 72 to 96 h after initial contact. However, upon penetration vascular continuity was established between parasite and host. In contrast, interactions with nonhosts provided evidence of active resistance mechanisms. Penetration of lettuce, marigold, and cowpea roots by S. asiatica was most frequently arrested in the cortex, and endophytic cells were necrotic 72 h after inoculation. Some species-specific differences were observed in the reactions of nonhosts to penetration, although in their general nature the interactions with S. asiatica were similar.

Initial Cellular Interactions Between Thielaviopsis basicola and Tobacco Root Hairs.

ABSTRACT Cellular events that occur during the initial interactions between Thielaviopsis basicola and root hairs of tobacco (Nicotiana tabacum) were examined microscopically. Time-course documentation of the infection process indicated a dynamic interaction between T. basicola and the living host cell. Upon root hair contact and recognition, the vegetative apex of T. basicola rapidly differentiated to form infection structures, and the host cell responded cytologically. Penetration was achieved by threadlike hyphae that subsequently developed distal swellings, and intracellular hyphae of sickle-shaped morphology advanced from the distal swelling and colonized the cell. Streaming of the host cytoplasm became aggregated near the infection site prior to penetration and accumulated around the infecting hyphae as long as the host cell was viable. Substantial callose deposition, in the form of a bell-shaped collar around infection structures, resulted from the cytological activity at the infection site. Penetration of dead root hairs was common, but did not lead to the development of infection structures or to a sustained association with the host tissue; T. basicola exited dead root hairs and resumed vegetative growth. The establishment of the parasitic relationship by T. basicola was characteristic of hemibiotrophic fungi in that, initially, biotrophic infection led to tissue colonization, and host cell survival was limited under parasitism.

Reassessment of the Role of Saprophytic Activity in the Ecology of Thielaviopsis basicola.

ABSTRACT The ability of Thielaviopsis basicola to survive saprophytically in soil was investigated using root tissue from susceptible hosts as organic substrates. Inoculum densities were lower in soils amended with root tissue than in nonamended controls after 2 and 4 weeks of incubation. The greatest decrease occurred in soils containing the highest concentration of root tissue or in soils in which root tissue included the soluble components of the living root. Reproduction by T. basicola also was examined in axenic media containing either killed root pieces or various carbohydrates as the sole carbohydrate source. T. basicola utilized killed root tissue as a carbohydrate source in axenic media, particularly in cultures in which root tissue included the soluble components. Enzymatic activities of T. basicola, however, did not result in maceration of the root tissue. T. basicola utilized sucrose and cellobiose, but did not utilize structural carbohydrates such as cellulose, hemicellulose, or pectin. Based on the absence of significant saprophytic ability, T. basicola should be classified ecologically as an obligate parasite.

Fine needle aspiration cytology in the management of palpable benign and malignant breast disease. Correlation with clinical and mammographic findings.

Two surgeons in a health maintenance organization group practice performed 280 fine needle aspirations (FNAs) on 257 palpable breast lesions in 200 patients. The cytology was interpreted by four pathologists at a community hospital. FNA cytology had a sensitivity of 96% and a specificity of 94% in these cases; there were no false-positive diagnoses of cancer. FNA cytology improved the identification of benign disease and decreased the risk of missing cancers. FNA cytology also improved the predictive value of mammographic information and was more helpful than mammography in demonstrating the need for biopsy of small palpable lesions that turned out to be "minimal" breast cancers.